Resorbable Calcium Phosphate Based Biopolymer-Cross-Linked Bone Replacement Material

ABSTRACT

A resorbable bone replacement material made of calcium phosphate particles of different phases which are embedded in an inventive-specific cross-linked collagen matrix. The goal is to form a non-brittle, bone replacement moulded body having a positive fit, i.e. having a shape which is anatomic and/or corresponds to the defect, which perfectly fills the bone defect and can be resorbed thereby. Said goal is achieved by producing the bone replacement material made of a mixture of calcium phosphate particles which is embedded in an inventive cross-linked collagen matrix. In particular, the collagen cross-linking is achieved by a Laccase-induced peptide cross-linking and suitable bridge molecules. Essentially substituted dihydroxyarmotes and/or substrates of the lignolytic polyphenoloxidases, such as Laccases, are suitable as bridge molecules. Also, monocyclic ortho-dihydroxyaromates, monocyclic para-dihydroxyaromates, bicyclic monohydroxyaromates, polycyclic monohydroxyaromates, bicyclic dihydroxyaromates, polycyclic dihydroxyaromates, bicyclic trihydroxyaromates, polycyclic trihydroxyaromates, or mixtures thereof are used. The inventive hydroxyaromates are not part of a polymer chain as opposed to the known conchal adhesive.

TECHNICAL FIELD

The invention relates to a resorbable bone substituent material made ofcalcium phosphate particles of different phases which are embedded in acollagen matrix cross-linked being specific to the invention.

BACKGROUND ART

Bone defects which cannot be closed in natural healing through theorganism require the application of bone substituent materials which canbe of natural or artificial origins, and which will fill the defect andbe restructured to endogenous bone.

The so-called “golden standard” represents the utilization of autologousbones then, i.e. the patient will be removed a piece of bone from thesound area, e.g. from the iliac crest (crista iliaca), which is used tofill the defect. Having greater bone defects or an inappropriateautologous bone, it is also fallen back upon foreign bone material whichwill be reconditioned accordingly. The advantage of providing foreignbones in larger quantities is confronted with their disadvantage thatvarying reconditioning processes may result in different mechanicalproperties (Palmer S. H., Gibbons C. L. M. H., Athanasou N. A.: JBJS(Br) 1999, 81-B, 333-5). Moreover, with foreign bones infections cannotbe excluded (Boyce, T., Edwards J., Scarborough N.: Orthop. Clin. NorthAm. 1999, 30(4), 571-81). For the application of a debiologized animalbone, the same objections apply as for the application of foreign bonessuch that various synthetic bone substituents have been developed.

The advantages of the synthetic bone substituents are in that checkingthe chemical composition and structure exists in addition to theavailability, and influencing the biologically effective properties canbe carried out such that an optimum course of therapy is achieved.

The plurality of the synthetic bone substituents comprise calciumminerals such as calcium carbonate, calcium sulphate and differentcalcium phosphates. In the past, particularly calcium phosphates such asβ-tricalcium phosphate and hydroxylapatite have been used, whereinhydroxylapatite is an essential constituent of the natural bone(Dorozhkin S. V., Epple M.: Angew. Chemie 2002, 114, 3260-77).

Calcium phosphate based bone substituent materials allow to be made invarious embodiments for different ranges of application. Granulates areused for dental bone defect filling, e.g., whereas injectable cementsare used with the stabilization of vertebral bodies. Load-carryingmoulded ceramic articles are inserted with defects of the skull and thegreat long bones.

In the load-carrying case, particular difficulties arise for bonesubstituent materials made of calcium phosphates. The necessarystructure is allowed to carry defined maximum forces only, and thenaturally available brittleness of synthetic calcium phosphates has tobe compensated by an adapted producing process. The increase of strengthis achieved, e.g. by sintering. Disadvantageously, the sintered calciumphosphates are reabsorbed then substantially more slowly than it wouldcorrespond to the normal course of therapy (LeGeros R. Z.: ClinicalMaterials 1993, 14, 65-88). Moreover, with sintering loss ofnanoporosity is notched up.

Another possibility to increase the strength of calcium phosphate basedbone substituent materials is compression by cold isostatic pressing.However, the porosity of these mouldings then gets largely lost suchthat, for compensating this effect, macroscopic structuring has to beprovided by mechanical remachining, e.g. bores (Tadic D., Epple M.:Biomaterials 2003, 24, 4565-71).

If the mechanical stressability does not play the important part, thencalcium phosphate based bone substituents can be produced with furtherprocesses permitting a better adjustment of the porosity. If oneconfines to inorganic components as material constituents, then inparticular sol-gel processes are just right to generate open-porednetwork structures (Brinker C J., Scherer G. W.: Sol-Gel Science: ThePhysics and Chemistry of Sol-Gel Processing, Academic Press, 1990).

In addition, if organic components are used as binding agents, thenmouldings are allowed to be generated both in immediately shapingprocesses (Michnaa S., Wua W., Lewis J. A.: Biomaterials 2005, 26,5632-9), e.g. as a three-dimensional grid pattern, and produced by pressforming (Weihe S., Wehmöller M., Tschakaloff A., von Oepen R., SchillerC., Epple M., Eufinger H.: Mund-, Kiefer-, Gesichtschirurgie 2001, 5:299-304). The porosity of these mouldings has to be set by the shapingprocess itself or by mechanical machining (D. Tadic D., M. Epple M.:Biomaterials 2003, 24, 4565-71).

Through the application of bone substituents which comprise adjustablecomponents of calcium phosphates having different solubilities withinthe body fluid and thus various resorbence rates, the course ofresorbence can be configured in a defined manner. As a result, thestrain is additionally supported by the bone substituent material over atime period, in which new bone tissue is developing and solidifying. Thecalcium phosphates with less solubility will be completely reabsorbedlater during remodelling of the bone (LeGeros R. Z.: Clinical Materials1993, 14, 65-88).

In summary, it is to be noted that calcium phosphate based bonesubstituent materials are successfully used for the treatment of simpledefects. However, at the moment application with heavy defects is notcarried out due to of material-related problems. These problems aboveall rest in that the actual processes are not able to meet substantialrequirements on bone substituent material and defect filling materialbodies formed from it, respectively:

(1) on the one hand, to produce positive bone substitute mouldings, i.e.shaped anatomically and according to the defect, respectively, whichcompletely fill the bone defect perfectly and non-positive as well, and

(2) on the other hand, to implement an easily resorbable structure.

Moreover, with the application of the actual manufacturing processes itis very difficult to fit into the bone substituent material additionalorganic or inorganic matters having strongly varying concentrations aswell as to provide an improvement of the load-carrying properties bymeans of embedded, permanent and resorbable supporting structures,respectively.

DISCLOSURE OF THE INVENTION

The problem formulation according to the invention is producing apositive non-brittle bone substitute moulding, i.e. being shapedanatomically and according to the defect, respectively, which completelyfills the bone defect perfectly and which is resorbable as well.

The solution of the problem formulation is achieved by producing thebone substituent from a mixture of calcium phosphate particles embeddedinto a collagen matrix being cross-linked according to the invention.

In particular, collagen cross-linking is achieved by laccase-inducedpeptide cross-linking and suitable bridging molecules.

As bridging molecules the substituted dihydroxy aromatics and/orsubstrates of lygnolytical polyphenol oxidases such as laccases aregenerally suitable.

Therefore, monocyclic orthodihydroxy aromatics, monocyclic paradihydroxyaromatics, bicyclic monohydro aromatics, polycyclic monohydroxyaromatics, bicyclic dihydroxy aromatics, polycyclic dihydroxy aromatics,bicyclic trihydroxy aromatics, polycyclic trihydroxy aromatics ormixtures thereof are used. In contrast to the well-known mussel glues,the hydroxyl aromatics according to the invention are not part of apolymer chain.

These aromatics may be further substituted. Preferred functional groupsare substituents selected from the group consisting of halogen, sulfo,sulfone, sulfamindo, sulfanyl, amino, amido, azo, imino and hydroxy.Then, it is to ascertain that substituted aromatics, in particularsubstituted dihydroxy aromatics, have very favourable polymerizationproperties such as fast polymerization, low natural linkage and goodstrength of cross-linking. Appropriate substitution of the aromaticsresults in that monohydroxy aromatics are also suitable as bridgingmolecule for cross-linking. Within the scope of this invention,substitution means that in addition to the hydroxyl groups 1, 2, 3 or 4further groups are bonded to the aromatics. Further, monohydroxylatedbiaryl compounds are also suited as bridging molecules.

Particularly preferred as a bridging molecule are phenol derivates whichcomprise a hydroxyl group or a methoxy group corresponding to theformulas 1 and 2, on the ortho position or para position,

wherein

n=0-10, preferably 0 or 1, in particular 0;R₁═OH or NH₂ or hal, preferably OH, Cl or Br, in particular OH;R₂═H, CH₃, CHO, COCH₃, CONH₂, CON-alkyl, CON-alkyl-OH, COOH, COO-alkyl,alkyl, substituted aromatic, in particular CON-alkyl or COO-alkyl, andR₃═H, CH₃, alkyl, substituted aromatic, in particular H or CH₃.

Herein, alkyl means branched or non-branched aliphatic hydrocarbonchains having preferably 1 to 20 carbon atoms, more preferably 1 to 6carbons, e.g. methyl, ethyl propyl, butyl, isobutyl, n-pentyl, n-hexyl.

Possible bridging molecules furthermore are compounds of formula 1 andof them the hydroquinone, which may be further substituted. Seen from apoint of view of fast bonding reaction, if possible, substituteddihydroxy aromatics having a low natural linkage are particularlyappropriate according to the invention. 2,5 dihydroxybenzamides arepreferably used, wherein 2,5-dihydroxy-N-2-hydroxyethylbenzamide isparticularly preferred.

In the case of aromatic trihydroxy compounds it is preferred that thereare not more than two hydroxyl groups per unit of benzene. Particularlypreferred are polyphenyls, i.e. biphenyl or triphenyl of the followingformula 3:

wherein

n=0-10, preferably 0 or 1,and

R₁═H and R₂═OH or R₁═OH and R₂═H.

Then, the phenyls of formula 3 may be substituted, e.g., in the orthoposition into a hydroxyl group having CH₃, CHO, COCH₃, CONH₂, CON-alkyl,CON-alkyl-OH, COOH, COO-alkyl, alkyl, substituted aromatic in particularCON-alkyl or COO-alkyl and/or in the metha position into a hydroxylgroup having CH₃, alkyl, substituted aromatic in particular CH₃.

Cross-linking of collagen occurs according to the invention under theinfluence of polyphenol oxidases such as lignolytic polyphenol oxidases,in particular laccases (EC 1.10.3.2). Laccases are known ascross-linkers. They are allowed to arise from plants, mushrooms,bacteria or insects or be derived from natural enzymes. The laccases tobe used within the scope of this invention may be produced recombinantlyor can be cleaned up.

Examples thereof are laccases being extracted from the species ofaspergillus, neurospora, podospora, botrytis, collybia, fomes, lentinus,pleurotus, pycnoporus, pyricularia, trametes, rhizoctonia, coprinus,psatyrella, myceliophthora, schtalidium, polyporus, phlebia or coriolus.The preparation of laccases is disclosed in EP 0947142.

By the application of polyphenol oxidases such as lignolytic polyphenoloxidases, preferably laccase (EC 1.10.3.2), the substrate spectrumthereof can be employed for the cross-linking reaction. Therefore, theinvention particularly distinguishes in that a wide range of bridgingmolecules can be used for cross-linking.

Variations of the concentrations approximately of 1 to 50 mM arepossible both with the individual component collagen and the bridgingmolecule matter.

Then, it must be considered, that according to the selected bridgingmolecule, an interfering natural reaction of the bridging molecule takesplace decreasing the formation of cross-linking bonds. An excessivelylow concentration of the bridging molecules results in a too slowreaction, an excessively high concentration results in stronger sidereactions through natural linkage. The concentration of the polyphenoloxidase influences the reaction rate wherein, according to application,faster cross-linking or a longer processability of the combination canbe achieved by varying the concentration. A preferred volume ratio forcross-linking of soft tissues, for example, is implemented in theformulation of 8.5 nM of collagen, 12.5 mM of2,5-dihydroxy-N-2-hydroxyethylbenzamide, 0.32 U (156 nmol ml⁻¹ min⁻¹) ofpolyphenol oxidase.

Cross-linking of a collagen matrix by means of bridging molecules withembedding calcium phosphate particles of different phases andconcentrations into the matrix results in a solid material with thesuitability as bone substituent.

The polyphenol oxidase and the polyphenols are dissolved preferably inphosphate buffer such as calcium phosphate or sodium phosphate buffer orPBS. The consistency of the used components of laccase and polyphenol isfrom liquid to pasty. The viscosity of the used individual componentscan be varied by solvents. The concentration of the solvents influencescross-linking.

Cross-linking reaction preferably occurs at pH value of 5 to 7. Thereaction is allowed to proceed within the temperature range of 2 to 80degrees centigrade, however, a temperature is preferred within the rangeof 20 to 37 degrees centigrade, in particular within the range of 25 to30 degrees centigrade.

In support of cross-linking another shorter-chain peptides can be used.In a particularly preferred embodiment of the invention about 50 percentof the amino acids of the peptide consist of lysine. Lysine and anotheramino acid may be arranged as a repeating dipeptide unit. Anothersuccession or absorbing of further amino acids of, in particulararginine, asparagine, glutamine or histidine (instead of lysine or inaddition thereto), serine or threonine (instead of tyrosine or inaddition thereto), of cysteine or other amino acids is also possible.

In a likewise preferred embodiment it exclusively concerns with polymersconsisting of two amino acids such as (lysine tyrosine)_(n), wherein ncan assume values of between 5 and 40 such as 5, 10 or 20.

Furthermore, (lysine tyrosine), is advantageously used as a peptide.With the use of (lysine tyrosine)_(n) within the matrix, lysine existsin a high concentration in the bone substituent material and has apositive effect on the proliferation and differentiation of bone cells.

Prior to cross-linking, the substance mixture has viscous properties andcan be arbitrarily shaped out. Then, simple shapes such as globules aswell as complex mouldings can be represented.

Furthermore, filling the porous mouldings with the viscous material andcuring thereof is possible.

In one preferred embodiment the material is configured as aninterconnectingly porous body.

Preferably, the part by weight to the total weight of calcium phosphateparticles is 70 to 95 percent, of collagen and oligopeptides is 5 to 15percent if necessary, and of the bridging molecule substance is about0.5 to 5 percent.

The bone substituent materials according to the invention are allowed toinclude further substances. In a preferred mode of implementation thematerial additionally includes SiO₂.

The bone substituent material can be doped with antibiotics. Then,antibiotics having free amino groups which have been mixed to the bonesubstituent prior to crosslinking will be bonded to collagen during thelaccase-induced cross-linking. On that occasion, it could bedemonstrated that the collagens doped in this manner with antibioticshave a strong antimicrobial effectiveness.

The bone substituent material can be inserted with and without asupporting structure. If a resorbable organic material is inserted assupporting structure, it is of particular advantage according to theinvention to select an organic material carrying free amino groups. Inthis case, cross-linking occurs by means of bridging molecules not onlytoward the collagen but toward the supporting structure as well suchthat a solid connection is obtained, however, which will be substitutedby endogenic tissue during the healing process. The mechanism ofresorbence can be in a hydrolytical or enzymatical manner. Inparticular, the peptides can be decomposed, and individual fragments canbe taken away and be separated. Alternatively, the fragments or aminoacids are also allowed to be incorporated into the regenerating tissue.

In a further embodiment, the porosity of the calcium phosphate mouldingis directed. Consequently, the mechanical properties of the resultingcomposite moulding are depending on the direction as well.

By cross-linking the contacting surfaces of two mouldings havingdirectionally depending mechanical properties which result in a solidconnection of the mouldings, the directional dependence of themechanical properties of the resulting moulding will be reduced.

By means of the solid combination of a plurality of two-dimensionalmouldings through cross-linking of their paired contacting surfaces, amulti-layer composite moulding is made which has a high mechanicalstressability.

WAY (S) FOR IMPLEMENTING THE INVENTION First Embodiment

A mixture from particles of different modifications of calcium phosphateand collagen is homogeneously mixed with 2,5dihydroxy-N-2-hydroxyethylbenzamide such that a concentration of 12.5 mMis achieved. Cross-linking is initiated by adding of laccase having anactivity of 0.32 U (156 mmol ml⁻¹ min⁻¹). After curing of the adhesivethe mineral constituents together with the adhesive material form,depending on the solid matter components, a high viscous or solidmaterial.

Second Embodiment

An experimental arrangement such as in the first embodiment is selected,however, in addition to the collagen an oligopeptide (length of 2 toapprox. 100 amino acids, preferably approx. 4 to appr. 20 amino acids)is added. Compared with usual proteinaceous amino acids, modified andatypical amino acids, respectively, such as hydroxylisine can also becomprised in the oligopeptide. Lysine containing oligopeptides arepreferably used, and it is of particular advantage when lysine isapproximately 50 percent of the amino acids of the peptide.Additionally, approximately 50 percent of the amino acids of the peptidemay be tyrosine. Lysine and tyrosine may be arranged, e.g. as arepeating dipeptide unit. In particular, by adding peptides whichconsisted of repeating dipeptide units of lysine and tyrosine([lys-tyr]_(n) or [tyr˜lys]n, n=5 or n=10, good cross-linking has beenachieved with the incorporation of the collagen and inclusion of thecalcium phosphates.

Third Embodiment

The oligopeptides described in the second embodiment are received in PBS(phosphate buffered saline, 2.7 M NaCl₂, 54 mM KCl, 87 mM Na₂HPO₄, 30 mMKH₂PO₄, pH 7.4) and laccase is added (Component 1).2,5-dihydroxy-N-2-hydroxyethyl-benzamide is dissolved in PBS (Component2). After combining both components, mixing with particles of differentmodifications of calcium phosphate and collagen is carried out. Thequantity of solvent is selected minimally in order to achieve a solutionof the components as concentrated as possible.

Fourth Embodiment

The viscous mixture of particles of different modifications of calciumphosphate and collagen, if necessary, and of further oligopeptidesaccording to the second embodiment 2,5-dihydroxy-N-2-hydroxyethylbenzamide and laccase, is inserted by means of capillary action,pressurizing or vacuum into the pores of an interconnectingly porousbrittle moulding prior to sintering based on calcium phosphate, whichafter curing forms a solid composite moulding in compound with the CaPmoulding. In a specific embodiment, the interconnectingly porousmoulding is formed such that the inner surface thereof is largelycovered with collagen being locally determined, cross-linked.

After cross-linking initiated by laccase, a material beinginterconnectingly porous with regard to both the mineral and thecollagen is obtained. When implanted, due to its osteoconductive actionthis material serves as a biological guide bar for the regeneration ofmissing bone and thus for healing the defect.

Fifth Embodiment

A composite moulding according to the third embodiment is prepared withthe specific feature that a simply open or multiply open hollow body ora closed hollow body is created by means of a multilayer cross-linkingof contacting surfaces. After the implantation the recovery of the bonedefect occurs while maintaining dimensional stability.

Sixth Embodiment

Preparation of a bone substituent according to the first embodiment withthe specific feature that an amino group carrying antibiotic has beenhomogeneously distributed in the mixture before adding of laccase. Aftercross-linking with the laccase the collagen is doped with theantibiotic. Thus, a bone substituent is formed which is activeingredient loaded both inside and on the surface.

Seventh Embodiment

Preparation of a multilayer composite moulding according to the sixthembodiment with the specific feature that identical or different activeingredients will be embedded into one or several layer boundaries.

1. A resorbable bone substituent material comprising calcium phosphateparticles of different phases and collagen, wherein the collagen is inthe form of a matrix that is at least partially cross-linked by asubstituted polyhydroxy aromatic compound under the action of a laccase.2. A resorbable bone substituent material according to claim 1, whereinthe substituted polyhydroxy aromatic compound is selected from the groupconsisting of: monocyclic dihydroxy aromatic compounds, bicyclicdihydroxy aromatic compounds, polycyclic dihydroxy aromatic compounds,bicyclic trihydroxy aromatic compounds, and polycyclic trihydroxyaromatic compounds.
 3. A resorbable bone substituent material accordingto claim 2, wherein the substituted polyhydroxy aromatic compound has amolecular weight of 110 to 1100 g/mol.
 4. A resorbable bone substituentmaterial according to claim 3, wherein the polyhydroxy aromatic is2,5-dihydroxy-N-2-hydroxyethyl benzamide.
 5. A bone substituent materialaccording to claim 1, the bone substituent material being in the form ofan interconnectingly porous body.
 6. A bone substituent materialaccording to claim 1, wherein the collagen matrix additionally comprisesa SiO₂ gel.
 7. A bone substituent material according to claim 5, whereinthe interconnectingly porous body has an inner surface and is formedsuch that the majority of the inner surface thereof is largely coveredby crosslinked collagen which is locally determined.
 8. A bonesubstituent material according to claim 1, wherein the various materialcomponents are arranged in an irregular sequence or regular sequence. 9.A bone substituent material according to claim 1 in the form of amoulding.
 10. A bone substituent material according to claim 9, furthercomprising an internal supporting structure.
 11. A bone substituentmaterial according to claim 10, wherein the supporting structureconsists of a metal.
 12. A bone substituent material according to claim11, wherein the metal is resorbable.
 13. A bone substituent materialaccording to claim 10, wherein the internal supporting structureconsists of resorbable organic material.
 14. A bone substituent materialaccording to claim 1 further comprising one or more active ingredientson either or both of external and internal surfaces.
 15. A bonesubstituent material according to claim 11, characterized in that saidactive ingredients will be introduced into the volume and will be justreleased through the resorbance of the material.
 16. A bone substituentmaterial according to claim 11, wherein said metal comprises titanium.